The present invention discloses the treatment of bacterial infections through the use of chemotherapeutic agent, antisense oligonucleotides. The invention also discloses the treatment of infections caused by bacteria such as Mycobacterium tuberculosis having resistance to one or more conventional chemotherapeutic agents.
The emergence of drug-resistant bacteria in general and drug-resistant tuberculosis in particular is rapidly becoming a major public health problem in the U.S. and miracle? Science 264: 360-365, 1994). The urgency in finding new treatment modalities for drug-resistant tuberculosis is emphasized by two recently published molecular and conventional epidemiological studies (Alland, D., et al. Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods. New Engl. J. Med. 330: 1710-1716, 1994; Small, P. M., et al. The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods. New Engl. J. Med., 330: 1703-1709, 1994). Not only was it demonstrated by utilization of DNA fingerprinting that more than a third of the newly diagnosed TB cases in New York and San Francisco are the result of recent person to person transmission, rather than activation of latent infections, but also that nearly half of the isolates from patients with recently transmitted infections in New York were drug-resistant M. tuberculosis. Of these drug-resistant isolates, half were resistant to multiple drugs. Drug-resistant M. tuberculosis strains, because of their ability to lead to a long infectious state, are especially active in the recent person to person transmissions.
Tuberculosis is the most widespread of human pathogenic diseases, having an annual number of new cases of active infection of 7.5 million cases and an annual number of deaths attributable to tuberculosis of 2.5 million worldwide according to the World Health Organization (Editorial, The Global Challenge of tuberculosis. The Lancet 344: 277-279, 1994).
Modern chemotherapy of tuberculosis began in 1952 with the development of isoniazid (isonicotinic acid hydrazide) which along with rifampin (or rifampicin) is still the mainstay of the treatment of tuberculosis. Streptomycin, which was shown to be effective in the treatment of tuberculosis before 1952, frequently resulted in treatment failure due to the rapidly developing resistance to streptomycin. Combinations of streptomycin with aminosalicylic acid and isoniazid were then utilized resulting in a high degree of cures in tuberculosis patients. The antituberculosis drugs that currently are being clinically utilized are isoniazid, rifampin, pyrazinamide and ethambutol as first line oral drugs; streptomycin, amikacin, kanamycin and capreomycin as injectable drugs; ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid and D-cycloserine as second-line oral drugs (Iseman, M. D. Treatment of multidrug-resistant tuberculosis. New England Journal of Medicine 329: 784-791, 1993; Mandell, G. L. and Sande, M. A. Antimicrobial agents, drugs used in the chemotherapy of tuberculosis and leprosy in The Pharmacological Basis of Therapeutics, Gilman A.G. et al. Editors, Pergamon Press, 1990, pp. 1146-1164).
Drug resistance and multidrug resistance in tuberculosis originates in spontaneous mutations which occur at predictable rates in the tubercle bacilli. The mutations are not linked and the rise of drug-resistant organisms is the result of these pre-existing mutations rather than a result of drug treatment or another novel mechanism not yet established or understood (Heym, B. et al. Implications of multidrug resistance for the future of short-course chemotherapy of tuberculosis: a molecular study. The Lancet 344: 293-298, 1994). Thus, the emergence of drug resistance is the direct result of the survival of random pre-existing mutations and the selection of the mutation-carrying organisms due to the killing of drug-sensitive organisms by effective drugs. Drug resistance in tubercle bacilli does not involve mechanisms related to the existence of increased efflux of drugs through multidrug resistant (MDR) channels as is the case with parasites such as Plasmodium falciparum. 
Oligonucleotides containing a base sequence complementary to selected sequences present on mRNAs were shown during the last several years to inhibit the synthesis of the specific proteins coded for by the targeted mRNAs. These oligonucleotides, known as antisense comprise a new class of therapeutic agents and have been demonstrated to inhibit the replication and expression of human immunodeficiency virus (HIV), as well as other viruses or cellular proteins in in vitro or in vivo screening systems. The internucleoside phosphate group of oligodeoxynucleotides can be chemically modified, for example, to methylphosphonates, phosphorothioatea, or phosphoroamidates in order to increase nuclease resistance of the modified oligonucleotide, without serious effects on its base sequence-specific hybridization to target mRNAs. Such modifications can afford reductions in the 50% inhibitory concentrations (IC50) to the 10xe2x88x927 M range.
U.S. Pat. No. 4,806,463 entitled xe2x80x9cInhibition of HTLV-III by exogenous oligonucleotidesxe2x80x9d to Goodchild and Zamecnik disclosed for the first time the use of modified oligonucleotides including oligodeoxynucleotide phosphorothioates which are oligonucleotides modified on the internal phosphate groups, for inhibiting the replication and gene expression of HTLV-III virus now called Human Immunodeficiency Virus (HIV). U.S. Pat. No. 5,276,019 entitled xe2x80x9cInhibitors for replication of retroviruses and for the expression of oncogene productsxe2x80x9d to Cohen et al. also discloses the use of modified oligodeoxynucleotide phosphorothioates for inhibiting viral replication in a host as well as inhibiting the replication of a human immunodeficiency virus replication in particular. The disclosures of these two patents are incorporated herein by reference.
Oligonucleotide drugs inhibit the expression of specific genes by three potential mechanisms. For RNA targets, at least two mechanisms of inhibition can be envisioned. Oligodeoxynucleotide hybridization to the complementary RNA sequences may inhibit the processing, nuclear export, or translation of mRNA by blocking the access of functional machinery to requisite mRNA sequences. Alternatively, oligodeoxynucleotide hybridization may lead to RNA cleavage by means of RNase H activities, which are DNA-RNA hybrid-dependent ribonucleases present in all cells examined. Evidence for both RNase H-dependent and independent modes of action has been presented for mRNA translational inhibition by complementary oligodeoxynucleotides in cell-free systems. Oligonucleotides can also be targeted to specific sequences of the DNA double helix where they inhibit transcription of specific genes (antigen or triplex strategy). The development of oligonucleotides and their modifications as therapeutic agents has been accelerated by recent advances in synthetic oligonucleotide chemistry.
Experimental evidence to establish that antisense technology can be extended to treating bacterial infections has not before existed to the knowledge of the inventors. If further has been unknown whether the mechanism of drug resistance in Mycobacterium tuberculosis would act to exclude oligonucleotides introduced exogenously. Finally, it was unknown whether antisense oligonucleotides would gain access through the bacterial cell wall if introduced exogenously, even for nonresistant strains.
The present invention discloses the use of antisense oligonucleotides for inhibiting bacterial growth and thus for the treatment of bacterial infections. Since bacterial infection previously was treated by a cheap, effective and readily available assortment of antibacterial agents, a significant aspect of the present invention is the disclosure that growth of bacteria resistant to one or more of the conventional therapeutic agents nonetheless can be readily inhibited by antisense oligonucleotides. The present invention demonstrates that conventional mechanisms which yield drug-resistant bacteria do not operate to exclude oligonucleotides and that antisense oligonucleotides can circumvent drug-resistance in bacteria.
According to one aspect of the invention, a method for treating an infection caused by a bacterium is provided. The method involves administering to a subject, preferably a human, in need of such treatment an effective amount of an antisense oligonucleotide that inhibits the growth of the bacterium. Useful oligonucleotides include oligoribonucleotides, oligodeoxyribonucleotides and modified versions of the same. Modified versions include those with modified internucleoside linkages such as phosphorothioate linkages, methylphosphonate linkages and phosphoroamidate linkages. Phosphorothioates are preferred. The oligonucleotides also may be chemically modified at either or both ends to prevent nucleolytic degradation. The oligonucleotides are constructed and arranged such that they hybridize under physiological conditions to a bacterial gene, the expression of which is essential for bacterial metabolism and growth. The invention is not limited to treatment of any particular bacterial infection, although preferred is treatment of infection by drug resistant strains of bacteria.
Various methods are provided for delivering the oligonucleotides of the invention in effective amounts. In one aspect of the invention, the subject is simultaneously treated with a compound such as ethambutol, which labilizes the bacterial wall and facilitates the delivery of the oligonucleotide into the bacterium. In another aspect of the invention, the oligonucleotides may be linked to a molecule that enhances bacterial wall transport. For example, the oligonucleotides may be covalently linked to a ligand for a receptor on a bacterium. Examples of ligands include D-cycloserine, D-glucosamine and biotin.
According to other aspects of the invention, articles of manufacture are contemplated. For example, antibacterial cocktails may be prepared which include a labilizing agent and the oligonucleotides of the invention. As another example, the invention provides an antibacterial agent that is an oligonucleotide of the invention covalently coupled to a molecule that enhances transport of the oligonucleotide across the bacterial cell wall. Such a molecule can be a ligand for a receptor on a bacterium. Specific examples include antisense oligonucleotides covalently coupled to D-cycloserine, D-glucosamine or biotin.